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The N-terminal PIN domain of the exosome subunit Rrp44 harbors endonuclease activity and tethers Rrp44 to the yeast core exosome.

Schneider C, Leung E, Brown J, Tollervey D - Nucleic Acids Res. (2009)

Bottom Line: Rrp44 lacking both exonuclease and endonuclease activity failed to support growth in strains depleted of endogenous Rrp44.Strains expressing Rrp44-exo and Rrp44-endo-exo exhibited different RNA processing patterns in vivo suggesting Rrp44-dependent endonucleolytic cleavages in the 5'-ETS and ITS2 regions of the pre-rRNA.Finally, the N-terminal PIN domain was shown to be necessary and sufficient for association with the core exosome, indicating its dual function as a nuclease and structural element.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, UK.

ABSTRACT
Nuclear and cytoplasmic forms of the yeast exosome share 10 components, of which only Rrp44/Dis3 is believed to possess 3' exonuclease activity. We report that expression only of Rrp44 lacking 3'-exonuclease activity (Rrp44-exo) supports growth in S288c-related strains (BY4741). In BY4741, rrp44-exo was synthetic-lethal with loss of the cytoplasmic 5'-exonuclease Xrn1, indicating block of mRNA turnover, but not with loss of the nuclear 3'-exonuclease Rrp6. The RNA processing phenotype of rrp44-exo was milder than that seen on Rrp44 depletion, indicating that Rrp44-exo retains important functions. Recombinant Rrp44 was shown to possess manganese-dependent endonuclease activity in vitro that was abolished by four point mutations in the putative metal binding residues of its N-terminal PIN domain. Rrp44 lacking both exonuclease and endonuclease activity failed to support growth in strains depleted of endogenous Rrp44. Strains expressing Rrp44-exo and Rrp44-endo-exo exhibited different RNA processing patterns in vivo suggesting Rrp44-dependent endonucleolytic cleavages in the 5'-ETS and ITS2 regions of the pre-rRNA. Finally, the N-terminal PIN domain was shown to be necessary and sufficient for association with the core exosome, indicating its dual function as a nuclease and structural element.

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The N-terminal PIN domain in Rrp44 harbors endonuclease activity in vivo. (A) Northern analysis of pre-rRNA processing in the GAL::rrp44 strain transformed with plasmids expressing either WT or mutant Rrp44 protein, or an empty vector pRS315. The mutants analyzed are rrp44-exo, rrp44-endo and rrp44-endo–exo (see Figure 5). RNA was isolated from GAL::rrp44 strains grown at 30°C under permissive conditions (GAL) and 10 h after transcriptional repression (GLU). RNA was separated on an 8% polyacrylamide/8 M urea gel and analyzed as described in Figure 1D. (B) Northern analysis of pre-rRNA processing in rrp44Δ (lanes 1–3) or rrp44Δrrp6Δ (lanes 4–6) strains transformed with a plasmid expressing either WT or mutant Rrp44 protein. Strains were grown at 30°C (rrp44Δ) or 25°C (rrp44Δ rrp6Δ) and RNA was analyzed as in (A). Two different exposures are shown for the probe recognizing precursors of the 5.8S rRNA (#020).
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Figure 6: The N-terminal PIN domain in Rrp44 harbors endonuclease activity in vivo. (A) Northern analysis of pre-rRNA processing in the GAL::rrp44 strain transformed with plasmids expressing either WT or mutant Rrp44 protein, or an empty vector pRS315. The mutants analyzed are rrp44-exo, rrp44-endo and rrp44-endo–exo (see Figure 5). RNA was isolated from GAL::rrp44 strains grown at 30°C under permissive conditions (GAL) and 10 h after transcriptional repression (GLU). RNA was separated on an 8% polyacrylamide/8 M urea gel and analyzed as described in Figure 1D. (B) Northern analysis of pre-rRNA processing in rrp44Δ (lanes 1–3) or rrp44Δrrp6Δ (lanes 4–6) strains transformed with a plasmid expressing either WT or mutant Rrp44 protein. Strains were grown at 30°C (rrp44Δ) or 25°C (rrp44Δ rrp6Δ) and RNA was analyzed as in (A). Two different exposures are shown for the probe recognizing precursors of the 5.8S rRNA (#020).

Mentions: Growth and handling of Saccharomyces cerevisiae were by standard techniques. Strains were grown at 25°C or 30°C in YPD or synthetic dropout medium containing 0.67% nitrogen base (Difco) and either 2% glucose or 2% galactose. Yeast RNA extraction and northern hybridization were performed as described (13). Northern signals were generally visualized by autoradiography, with the exception of the lighter exposure in Figure 6B, which was generated by a Fuji FLA-5100 PhosphorImager. Oligonucleotide probes are listed in Table S1.Figure 6.


The N-terminal PIN domain of the exosome subunit Rrp44 harbors endonuclease activity and tethers Rrp44 to the yeast core exosome.

Schneider C, Leung E, Brown J, Tollervey D - Nucleic Acids Res. (2009)

The N-terminal PIN domain in Rrp44 harbors endonuclease activity in vivo. (A) Northern analysis of pre-rRNA processing in the GAL::rrp44 strain transformed with plasmids expressing either WT or mutant Rrp44 protein, or an empty vector pRS315. The mutants analyzed are rrp44-exo, rrp44-endo and rrp44-endo–exo (see Figure 5). RNA was isolated from GAL::rrp44 strains grown at 30°C under permissive conditions (GAL) and 10 h after transcriptional repression (GLU). RNA was separated on an 8% polyacrylamide/8 M urea gel and analyzed as described in Figure 1D. (B) Northern analysis of pre-rRNA processing in rrp44Δ (lanes 1–3) or rrp44Δrrp6Δ (lanes 4–6) strains transformed with a plasmid expressing either WT or mutant Rrp44 protein. Strains were grown at 30°C (rrp44Δ) or 25°C (rrp44Δ rrp6Δ) and RNA was analyzed as in (A). Two different exposures are shown for the probe recognizing precursors of the 5.8S rRNA (#020).
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Figure 6: The N-terminal PIN domain in Rrp44 harbors endonuclease activity in vivo. (A) Northern analysis of pre-rRNA processing in the GAL::rrp44 strain transformed with plasmids expressing either WT or mutant Rrp44 protein, or an empty vector pRS315. The mutants analyzed are rrp44-exo, rrp44-endo and rrp44-endo–exo (see Figure 5). RNA was isolated from GAL::rrp44 strains grown at 30°C under permissive conditions (GAL) and 10 h after transcriptional repression (GLU). RNA was separated on an 8% polyacrylamide/8 M urea gel and analyzed as described in Figure 1D. (B) Northern analysis of pre-rRNA processing in rrp44Δ (lanes 1–3) or rrp44Δrrp6Δ (lanes 4–6) strains transformed with a plasmid expressing either WT or mutant Rrp44 protein. Strains were grown at 30°C (rrp44Δ) or 25°C (rrp44Δ rrp6Δ) and RNA was analyzed as in (A). Two different exposures are shown for the probe recognizing precursors of the 5.8S rRNA (#020).
Mentions: Growth and handling of Saccharomyces cerevisiae were by standard techniques. Strains were grown at 25°C or 30°C in YPD or synthetic dropout medium containing 0.67% nitrogen base (Difco) and either 2% glucose or 2% galactose. Yeast RNA extraction and northern hybridization were performed as described (13). Northern signals were generally visualized by autoradiography, with the exception of the lighter exposure in Figure 6B, which was generated by a Fuji FLA-5100 PhosphorImager. Oligonucleotide probes are listed in Table S1.Figure 6.

Bottom Line: Rrp44 lacking both exonuclease and endonuclease activity failed to support growth in strains depleted of endogenous Rrp44.Strains expressing Rrp44-exo and Rrp44-endo-exo exhibited different RNA processing patterns in vivo suggesting Rrp44-dependent endonucleolytic cleavages in the 5'-ETS and ITS2 regions of the pre-rRNA.Finally, the N-terminal PIN domain was shown to be necessary and sufficient for association with the core exosome, indicating its dual function as a nuclease and structural element.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, UK.

ABSTRACT
Nuclear and cytoplasmic forms of the yeast exosome share 10 components, of which only Rrp44/Dis3 is believed to possess 3' exonuclease activity. We report that expression only of Rrp44 lacking 3'-exonuclease activity (Rrp44-exo) supports growth in S288c-related strains (BY4741). In BY4741, rrp44-exo was synthetic-lethal with loss of the cytoplasmic 5'-exonuclease Xrn1, indicating block of mRNA turnover, but not with loss of the nuclear 3'-exonuclease Rrp6. The RNA processing phenotype of rrp44-exo was milder than that seen on Rrp44 depletion, indicating that Rrp44-exo retains important functions. Recombinant Rrp44 was shown to possess manganese-dependent endonuclease activity in vitro that was abolished by four point mutations in the putative metal binding residues of its N-terminal PIN domain. Rrp44 lacking both exonuclease and endonuclease activity failed to support growth in strains depleted of endogenous Rrp44. Strains expressing Rrp44-exo and Rrp44-endo-exo exhibited different RNA processing patterns in vivo suggesting Rrp44-dependent endonucleolytic cleavages in the 5'-ETS and ITS2 regions of the pre-rRNA. Finally, the N-terminal PIN domain was shown to be necessary and sufficient for association with the core exosome, indicating its dual function as a nuclease and structural element.

Show MeSH
Related in: MedlinePlus